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Plasmacytoid dendritic cell bactericidal activity against Burkholderia pseudomallei
Microbes and Infection 17 (2015) 311e316 www.elsevier.com/locate/micinf
Short communication
Plasmacytoid dendritic cell bactericidal activity against Burkholderia pseudomallei Natasha L. Williams*, Jodie L. Morris, Catherine M. Rush, Natkunam Ketheesan Australian Institute of Tropical Health and Medicine, College of Public Health, Medicine and Veterinary Sciences, James Cook University, Townsville, Queensland 4811, Australia Received 28 February 2014; accepted 11 December 2014 Available online 19 December 2014
Abstract Melioidosis sepsis, caused by Burkholderia pseudomallei, is associated with high mortality due to an overwhelming inflammatory response. Plasmacytoid dendritic cells (pDC) are potent producers of type I interferons (IFN). This study investigated whether pDC and type I IFN play a role during the early stages of B. pseudomallei infection. Human and murine pDC internalised and killed B. pseudomallei as efficiently as murine conventional DC (cDC). pDC derived from B. pseudomallei-susceptible (BALB/c) mice demonstrated poor intracellular killing and increased IFN-alpha compared to pDC derived from B. pseudomallei-resistant (C57BL/6) mice. This is the first evidence of pDC bactericidal activity against B. pseudomallei infection. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Burkholderia pseudomallei; Melioidosis; Dendritic cells; Interferon-alpha; Interferon-beta
1. Introduction In acute cases of melioidosis, a bacterial infection caused by Burkholderia pseudomallei, patients can rapidly develop fatal septic shock [1]. Characteristics of melioidosis sepsis include significantly increased proinflammatory cytokine expression and the development of multiple organ failure [2]. Investigations of immune cell interactions with B. pseudomallei have found that the macrophage and neutrophil provide important innate defence against B. pseudomallei, while the conventional dendritic cell (cDC) provides a link between the innate and adaptive immune responses [3e10]. To our knowledge, there have been no reports on the role of plasmacytoid DC (pDC) in the immune response toward B. pseudomallei infection.
The capacity to produce large quantities of type I interferons (IFN) is one feature that sets pDC apart from cDC [11]. Historically, pDC and type I IFN were considered ‘antiviral’ mechanisms. It has now been recognised that pDC and type I IFN are capable of eliciting anti-bacterial activity that can be beneficial or detrimental to the host [12e15]. The contribution of pDC to clearance or persistence of B. pseudomallei infection has not been investigated. Therefore, using human and murine pDC, the purpose of this study was to assess the ability of pDC to internalise and kill B. pseudomallei, quantify the type I IFN response of pDC toward B. pseudomallei and determine if B. pseudomallei stimulates phenotypic maturation of pDC.
2. Materials and methods * Corresponding author. Australian Institute of Tropical Health and Medicine, College of Public Health, Medicine and Veterinary Sciences, James Cook University, 1 Solander Road, Townsville, Queensland 4811, Australia. Tel.: þ61 7 4781 6646; fax: þ61 7 4779 1526. E-mail address: [email protected] (N.L. Williams).
2.1. Enrichment of human pDC from peripheral blood Plasmacytoid DC were enriched from peripheral blood mononuclear cells (PBMC), of four healthy male individuals
http://dx.doi.org/10.1016/j.micinf.2014.12.007 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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(aged 28e52), using a pDC isolation kit (Stemcell EasySep™) according to manufacturer's instructions. Prior informed consent was received from all study participants. James Cook University Human Ethics Committee (H4470) provided institutional ethics approval. Purity of enriched pDC (hLMAX (lineage), HLA-DRþ, CD123þ) was confirmed by flow cytometry using a pDC identification kit (IMGENEX, Jomar Bioscience P/L). All flow cytometry was performed using a FACSCalibur with Cell Quest software (BD Biosciences) and post-acquisition analysis was performed using Flow Jo software (Tree Star Inc.). The purity of human pDC enriched from PBMC was 93.9 ± 2.1% (mean ± SEM; data not shown). 2.2. Culture of murine pDC from bone marrow supplemented with FLT-3L Bone marrow (BM) was isolated from C57BL/6 (B. pseudomallei-resistant) and BALB/c (B. pseudomallei-susceptible) mice (n ¼ 3 each, 8e12 week old, James Cook University Small Animal Breeding Facility). James Cook University Animal Ethics Committee (A1601) provided institutional ethics approval. BM cells were cultured at 1 106 cells/ml in pDC media (RPMI-1640 with 10% HIFBS, 1.5 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-Mercaptoethanol and 2.5 mmol/l HEPES buffer) supplemented with FLT-3L (150 ng/ml, Peprotech) using methods previously described [16]. On day 5, 50% of the culture media was replaced. On day 10, pDC expressing PDCA-1 were magnetically selected using biotinylated anti-PDCA-1 antibody (eBioscience) and streptavidin-magnetic nanoparticles (BD Biosciences). Enriched pDC, defined as PDCA-1þ/CD11cþ/B220þ/Ly6Cþ cells, were evaluated by flow cytometry using antimouse CD11c Biotin þ Streptavidin-APC (BD Biosciences), anti-mouse PDCA-1 PE (eBioscience), anti-mouse B220 PerCP-Cy5.5 (BD Biosciences) and anti-mouse Ly-6C FITC (BD Biosciences) [17]. The purity was 91 ± 2.8% for C57BL/6 pDC and 90 ± 3.2% for BALB/c pDC (mean ± SEM; data not shown). 2.3. Culture of murine cDC from BM supplemented with GM-CSF Bone marrow from C57BL/6 and BALB/c mice was also used to culture cDC according to published methods [6]. Isolated BM cells (2 105 cells/ml) were cultured in DC media (RPMI-1640 with 10% HI-FBS, 1.5 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mM 2Mercaptoethanol) supplemented with 10% supernatant from Ag8653 myeloma cells transfected with the gene encoding murine GM-CSF (kindly provided by B. Stockinger, MRC National Institute for Medical Research, Mill Hill, London, UK). On day 3 & 6, 50% of the media was replaced. On day 10 of culture, cDC were harvested.
2.4. B. pseudomallei infection of cells Human pDC (5 104), murine pDC (1 105) and cDC (1 105) were seeded into 96 well plates in pDC media and cultured at 37 C with 5% CO2. Two clinical B. pseudomallei isolates, NCTC 13178 (high virulence) and NCTC 13179 (low virulence; infection of murine pDC and cDC only), were grown to log phase then added to cells at a MOI 1:1 as previously described [6,18]. CpG ODN 2216 (3 mg/ml; SigmaeAldrich) was used as a positive stimulant [19]. After 4 h of culture, internalisation of B. pseudomallei by human pDC, murine pDC and murine cDC was assessed and kanamycin (250 mg/ml; SigmaeAldrich) added to parallel cultures to kill extracellular B. pseudomallei. After 24 h of culture, B. pseudomallei survival within murine pDC and cDC, type I IFN production by human and murine pDC and phenotypic maturation of murine pDC was assessed. 2.5. Assessment of internalisation and intracellular survival of B. pseudomallei Internalisation of B. pseudomallei by human pDC, murine pDC and murine cDC and the intracellular survival of B. pseudomallei within murine pDC and cDC was assessed as previously described [6]. The intracellular bacteria within pDC and cDC were released by lysing the cells with 0.1% Triton-X. Lysates were subsequently diluted and plated in triplicate on Ashdown agar and colonies enumerated after 48 h of culture at 37 C. The percentage of B. pseudomallei internalised at 4 h and the percentage of intracellular B. pseudomallei surviving at 24 h was determined. 2.6. Assessment of type I IFN production by pDC Cell culture supernatants from 24 h uninfected, B. pseudomallei-infected and CpG ODN 2216 -stimulated human and murine pDC were collected and stored at 80 C. The concentration of IFN-a and b in cell culture supernatants was quantified using human and mouse IFN-a (eBioscience) and IFN-b ELISA kits (Sapphire Bioscience) according to manufacturer's instructions. 2.7. Analysis of DC maturation markers on murine pDC Expression of MHC class II and CD86 on 24 h uninfected and B. pseudomallei-infected pDC was analysed by flow cytometry using anti-mouse PDCA-1 Biotin and StreptavidinPerCP (eBioscience), anti-mouse MHC class II PE (BD Biosciences), anti-mouse CD86 PE (BD Biosciences) [20]. The percentage of pDC that were PDCA-1þ/MHCclassIIþ and PDCA-1þ/CD86þ was determined. 2.8. Statistical analysis Statistical analysis of data was performed using GraphPad Prism 6 Software. The production of IFN-a and b by human
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pDC was compared using a 1way ANOVA with Tukey's posthoc multiple comparisons. Murine pDC response to stimulation was tested using 2way ANOVA with post-hoc multiple comparisons to enable comparison between mouse strains and stimulation groups. Differences between tested groups were considered significant if the P value 0.05. 3. Results 3.1. B. pseudomallei internalisation and type I IFN production by human pDC Internalisation of B. pseudomallei (NCTC 13178) by human pDC was evident after 4 h (2 ± 0.67%, mean ± SEM; data not shown). High levels of IFN-a and b were produced by pDC stimulated with CpG ODN 2216 (positive control; Fig. 1). However, B. pseudomallei-infected human pDC demonstrated low IFN-a and b production (Fig. 1). No significant difference was observed in the type I IFN response to B. pseudomallei compared to uninfected human pDC (Fig. 1). 3.2. B. pseudomallei internalisation and intracellular survival within murine pDC and cDC The percentage of B. pseudomallei (NCTC 13178, high virulence) internalised by C57BL/6 pDC was comparable to BALB/c pDC (Fig. 2A). When two B. pseudomallei isolates of contrasting high virulence (NCTC 13178) and low virulence (NCTC 13179) were compared, no significant difference in mean percentage of B. pseudomallei internalised was observed for C57BL/6 pDC (12.6 ± 3.2% and 8.5 ± 1.6% respectively; ±SEM, P ¼ 0.34; data not shown) or for BALB/c pDC (12.2 ± 2.3% and 10.5 ± 1.1% respectively; ±SEM, P ¼ 0.83; data not shown). The internalisation of B. pseudomallei (NCTC 13178) by pDC was also assessed in parallel with murine cDC and found to be comparable (Fig. 2A).
Fig. 1. Type I IFN production by human pDC The production of type I IFN by uninfected, CpG ODN 2216-stimulated and B. pseudomallei einfected (NCTC 13178, high virulence) human pDC was determined by quantifying IFN-a and b concentrations in culture supernatants after 24 h. Human pDC demonstrated high type I IFN production in response to positive stimulation with CpG ODN 2216 but low type I IFN production when infected with B. pseudomallei. Data points represent human pDC isolated from 4 individuals and the line bars depict the mean ± SEM. *P 0.05 and ** P 0.01 determined using a 2way ANOVA with post-hoc multiple comparisons.
Fig. 2. Burkholderia pseudomallei internalisation and intracellular survival within murine pDC and cDC Viable intracellular B. pseudomallei (NCTC 13178, high virulence) within murine pDC and cDC generated from C57BL/6 and BALB/c mice were enumerated at 4 and 24 h of co-culture. The percentage of A) B. pseudomallei internalised at 4 h and B) B. pseudomallei surviving at 24 h was calculated. A) Internalisation of B. pseudomallei was similar in both C57BL/6 and BALB/c pDC. B) The intracellular survival of B. pseudomallei was higher in BALB/c pDC compared to C57BL/6 pDC, though not statistically significant (P ¼ 0.06). This trend was significant for BALB/c cDC compared to C57BL/6 cDC. Bars depict mean ± SEM of three experiments, where three replicate samples were assessed in triplicate. *P 0.05 and NS e not significant determined using a 2way ANOVA with post-hoc multiple comparisons.
The intracellular survival of B. pseudomallei (NCTC 13178) was higher in BALB/c pDC compared to C57BL/6 pDC (Fig. 2B). Both murine pDC and cDC derived from BALB/c mice demonstrated a trend for reduced B. pseudomallei killing compared to pDC and cDC derived from C57BL/6 mice, this trend was statistically significant for BALB/c cDC compared to C57BL/6 cDC. Intracellular survival of B. pseudomallei in BALB/c cDC was also significantly higher than in BALB/c pDC (Fig. 2B). pDC demonstrated similar killing against intracellular B. pseudomallei of high (NCTC 13178) and low virulence (NCTC 13179); C57BL/6 pDC (0.6 ± 0.2% and 6.1 ± 2.6% mean B. pseudomallei survival ± SEM respectively; P ¼ 0.54; data not shown) and BALB/c pDC (6.3 ± 2.1% and 12.2 ± 7.8% mean B. pseudomallei survival ± SEM respectively; P ¼ 0.12; data not shown).
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3.3. Type I IFN production by murine pDC High IFN-a and b was observed for pDC stimulated with CpG ODN 2216 (positive control; Fig. 3A). However, BALB/c pDC demonstrated a reduced capacity to produce IFN-a
(Fig. 3A) and b (data not shown) compared to C57BL/6 pDC. Therefore, the increase in IFN-a/b above the baseline (uninfected pDC) was used for comparisons. The increase in IFN-a by B. pseudomallei-infected BALB/c pDC was significantly higher than that observed for B. pseudomallei-infected C57BL/6 pDC (Fig. 3B). IFN-a production by C57BL/6 and BALB/c pDC was comparable for B. pseudomallei isolates of high (NCTC 13178) and low (NCTC 13179) virulence. Although, the increase in IFN-b produced by C57BL/6 and BALB/c pDC in response to the high virulence B. pseudomallei isolate (NCTC 13178) was comparable, the low virulence B. pseudomallei isolate (NCTC 13179) stimulated significantly less IFN-b production by C57BL/6 pDC (Fig. 3C). 3.4. Murine pDC phenotypic maturation in response to B. pseudomallei To determine if exposure to B. pseudomallei activated phenotypic maturation of pDC, the expression of MHC class II and CD86 on uninfected and B. pseudomallei-infected pDC was compared. MHC class II expression decreased on C57BL/ 6 pDC (1.6 ± 1.5% mean change in MHC IIþ ± SEM) and BALB/c pDC (6.1 ± 3.1% mean change in MHC IIþ ± SEM) infected with B. pseudomallei (NCTC 13178; data not shown). Expression of CD86 increased on C57BL/6 pDC (0.3 ± 3.0% mean change in CD86þ ± SEM) and BALB/c pDC (6.1 ± 1.8% mean change in CD86þ ± SEM) infected with B. pseudomallei (NCTC 1378; data not shown). The changes observed were of a low magnitude and not significant due to high MHC class II and CD86 expression on uninfected murine pDC; 77% and 71% mean MHC IIþ and CD86þ respectively for C57BL/6 pDC, 70% and 72% mean MHC IIþ and CD86þ respectively for BALB/c pDC (data not shown). 4. Discussion
Fig. 3. Type I IFN production by murine pDC To investigate the type I IFN response of murine pDC derived from C57BL/6 and BALB/c mice, the concentration of IFN-a and b in culture supernatants was determined. A) Murine pDC from BALB/c mice, which are known to be poor producers of type I IFN, demonstrated a significantly reduced capacity to produce IFN-a and b (not shown) when stimulated with CpG ODN 2216 for 24 h. Therefore, to compare the type I IFN response of pDC toward B. pseudomallei, the concentration of type I IFN in culture supernatants of uninfected and B. pseudomallei-infected (NCTC 13178, high virulence) murine pDC was quantified and expressed as the fold increase in B) IFN-a and C) IFN-b above baseline (uninfected pDC). B) The increase in IFN-a was significantly higher in B. pseudomallei-infected BALB/c pDC compared to B. pseudomallei-infected C57BL/6 pDC and was not influenced by isolate virulence. C) The production of IFN-b by C57BL/6 pDC was similar to BALB/c pDC in response to NCTC 13178 (high virulence). However, the low virulence B. pseudomallei isolate (NCTC 13179) stimulated significantly less IFN-b production by C57BL/6 pDC. Bars depict mean ± SEM of a representative experiment of three repeat experiments, where three replicate samples were assessed in duplicate. *P 0.05 and **P 0.01 determined using a 2way ANOVA with post-hoc multiple comparisons.
The role of pDC during bacterial infections is underestimated and often thought of as immunomodulating due to conflicting evidence for pDC bactericidal and antigen presenting capacity [21]. This is the first report of internalisation and intracellular killing of B. pseudomallei by both human and murine pDC. The bactericidal activity of murine pDC differed for cells derived from B. pseudomallei-susceptible and resistant hosts. Importantly, murine pDC were as efficient as murine cDC at internalising and killing intracellular B. pseudomallei, providing evidence for the potential of pDC in processing and presentation of B. pseudomallei antigens to T cells. The type I IFN response of pDC varies for different infections and has been implicated in driving an excessive cytokine response that is detrimental to the host [12]. In this study, the production of IFN-a and b by human and murine pDC infected with B. pseudomallei was low in comparison to cytokine levels in CpG ODN 2216 stimulated cultures. Contrasting type I IFN responses by pDC toward bacteria have been reported. Although pDC are capable of producing high
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type I IFN responses to bacteria such as Staphylococcus aureus, other bacteria such as Streptococcus pyogenes, stimulate low type I IFN responses in human pDC similar that observed for B. pseudomallei-infected human pDC [14,24]. Interestingly, B. pseudomallei isolates of high and low virulence induced comparable type I IFN responses. BALB/c mice are known to be poor IFN-a/b producers compared to C57BL/ 6 mice [22]. The increase in IFN-a production by B. pseudomallei-infected BALB/c pDC was significantly higher than that observed for B. pseudomallei-infected C57BL/6 pDC. This trend was observed for pDC infected with both B. pseudomallei isolates of high (NCTC 13178) and low (NCTC 13179) virulence. In contrast, significant differences in IFN-b production between BALB/c and C57BL/6 pDC in response B. pseudomallei was only observed for the low virulence isolate (NCTC 13179). The BALB/c mice are highly susceptible to B. pseudomallei infection and develop disease that parallels the acute form of human melioidosis [3]. The observation that significantly increased IFN-a production correlated with pDC from B. pseudomallei-susceptible mice suggests that the increased IFN-a may be detrimental to the host during B. pseudomallei infection. Following activation, pDC lose their plasmacytoid morphology and acquire morphology similar to cDC. Increased MHC class II and CD86 expression on mature pDC is important for activation of naive T cells [20]. In some cases, pDC maturation responses are exploited to develop an inappropriate immune response that benefits the persistence of bacteria [14]. In this study, B. pseudomallei infection of murine pDC resulted in distinct differences in MHC class II and CD86 expression on BALB/c pDC and C57BL/6 pDC. Unlike cDC which have been shown to significantly increase MHC class II and CD86 expression in response to B. pseudomallei, MHC class II expression was decreased on murine pDC [6]. The magnitude of these changes was masked by high baseline expression on uninfected murine pDC. FLT-3L supplementation for 10 days to improve pDC yield and manipulation of cells to positively select the PDCA-1 positive pDC are likely to have contributed to the elevated baseline expression of MHC class II and CD86 [23]. In summary, this is the first description of the functional responses of human and murine pDC toward B. pseudomallei. We demonstrated that pDC internalised and killed B. pseudomallei as efficiently as cDC. B. pseudomallei virulence did not correlate with an ability to exploit pDC for intracellular survival or affect type I IFN production. Differences in bactericidal activity and IFN-a production were observed for murine pDC generated from B. pseudomallei-susceptible BALB/c mice compared to B. pseudomallei-resistant C57BL/ 6 mice. The BALB/c mouse is a well described animal model of the acute form of human melioidosis [3]. Our findings implicate pDC as an additional cell-mediator contributing to the altered immune responses that underlie the host susceptibility toward B. pseudomallei infection. Further studies on the downstream responses of pDC and type I IFN signalling are required to elucidate their ability to modulate other innate immune cells and to activate B. pseudomallei-specific T cell
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responses during the early phases of B. pseudomallei infection. Conflicts of interest statement The authors declare no conflicts of interest. Acknowledgements We acknowledge the contribution of the study participants who donated their time and blood samples for isolating human plasmacytoid dendritic cells. The Far North Queensland Hospital Foundation and James Cook University supported this study. References [1] Currie BJ, Fisher DA, Howard DM, Burrow JNC, Lo D, Selvanayagam S, et al. Endemic melioidosis in tropical Northern Australia: a 10-year prospective study and review of the literature. Clin Infect Dis 2000;31:981e6. [2] Wiersinga W, Dessing M, Kager P, Cheng A, Limmathurotsakul D, Day N, et al. High-throughput mRNA profiling characterizes the expression of inflammatory molecules in sepsis caused by Burkholderia pseudomallei. Infect Immun 2007;75:3074e9. [3] Leakey AK, Ulett GC, Hirst RG. BALB/c and C57Bl/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis. Microb Pathog 1998;24:269e75. [4] Barnes JL, Williams NL, Ketheesan N. Susceptibility to Burkholderia pseudomallei is associated with host immune responses involving tumor necrosis factor receptor-1 (TNFR1) and TNF receptor-2 (TNFR2). FEMS Immunol Med Microbiol 2008;52:379e88. [5] Woodman ME, Worth RG, Wooten RM. Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils. PLoS ONE 2012;7:e52276. [6] Williams NL, Kloeze E, Govan BL, Korner H, Ketheesan N. Burkholderia pseudomallei enhances maturation of bone marrow-derived dendritic cells. Trans R Soc Trop Med Hyg 2008;102(Suppl 1):S71e5. [7] Charoensap J, Engering A, Utaisincharoen P, van Kooyk Y, Sirisinha S. Activation of human monocyte-derived dendritic cells by Burkholderia pseudomallei does not require binding to the C-type lectin DC-SIGN. Trans R Soc Trop Med Hyg 2008;102(Suppl 1):S76e81. [8] Horton RE, Morrison NA, Beacham IR, Peak IR. Interaction of Burkholderia pseudomallei and Burkholderia thailandensis with human monocyte-derived dendritic cells. J Med Microbiol 2012;61:607e14. [9] Hodgson KA, Morris JL, Feterl ML, Govan BL, Ketheesan N. Altered macrophage function is associated with severe Burkholderia pseudomallei infection in a murine model of type 2 diabetes. Microbes Infect 2011;13:1177e84. [10] Elvin SJ, Healey GD, Westwood A, Knight SC, Eyles JE, Williamson ED. Protection against heterologous Burkholderia pseudomallei strains by dendritic cell immunization. Infect Immun 2006;74:1706e11. [11] Reizis B, Bunin A, Ghosh HS, Lewis KL, Sisirak V. Plasmacytoid dendritic cells: recent progress and open questions. Annu Rev Immunol 2011;29:163e83. [12] Decker T, Muller M, Stockinger S. The yin and yang of type I interferon activity in bacterial infection,. Nat Rev Immunol 2005;5:675e87. [13] Schiavoni G, Mauri C, Carlei D, Belardelli F, Pastoris MC, Proietti E. Type I IFN protects permissive macrophages from Legionella pneumophila infection through an IFN-gamma-independent pathway. J Immunol 2004;173:1266e75.
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[14] Parcina M, Miranda-Garcia MA, Durlanik S, Ziegler S, Over B, Georg P, et al. Pathogen-triggered activation of plasmacytoid dendritic cells induces IL-10-producing B cells in response to Staphylococcus aureus. J Immunol 2013;190:1591e602. [15] Meyer-Wentrup F, Benitez-Ribas D, Tacken PJ, Punt CJ, Figdor CG, de Vries IJ, et al. Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-alpha production. Blood 2008;111:4245e53. [16] Brasel K, De Smedt T, Smith JL, Maliszewski CR. Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures. Blood 2000;96:3029e39. [17] Hochrein H, O'Keeffe M, Wagner H. Human and mouse plasmacytoid dendritic cells. Hum Immunol 2002;63:1103e10. [18] Barnes JL, Ketheesan N. Route of infection in melioidosis. Emerg Infect Dis 2005;11:638e9. [19] Krug A, Rothenfusser S, Hornung V, Jahrsdorfer B, Blackwell S, Ballas ZK, et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol 2001;31:2154e63.
[20] Mitchell LM, Brzoza-Lewis KL, Henry CJ, Grayson JM, Westcott MM, Hiltbold EM. Distinct responses of splenic dendritic cell subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo. Cell Immunol 2011;268:79e86. [21] Villadangos JA, Young L. Antigen-presentation properties of plasmacytoid dendritic cells. Immunity 2008;29:352e61. [22] Shankar AH, Morin P, Titus RG. Leishmania major: differential resistance to infection in C57BL/6 (high interferon-alpha/beta) and congenic B6.C-H-28c (low interferon-alpha/beta) mice. Exp Parasitol 1996;84:136e43. [23] Grauer O, Wohlleben G, Seubert S, Weishaupt A, Kampgen E, Gold R. Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique. Histochem Cell Biol 2002;117:351e62. [24] Veckman V, Julkunen I. Streptococcus pyogenes activates human plasmacytoid and myeloid dendritic cells. J Leukoc Bio 2008;83:296e304.
Short communication
Plasmacytoid dendritic cell bactericidal activity against Burkholderia pseudomallei Natasha L. Williams*, Jodie L. Morris, Catherine M. Rush, Natkunam Ketheesan Australian Institute of Tropical Health and Medicine, College of Public Health, Medicine and Veterinary Sciences, James Cook University, Townsville, Queensland 4811, Australia Received 28 February 2014; accepted 11 December 2014 Available online 19 December 2014
Abstract Melioidosis sepsis, caused by Burkholderia pseudomallei, is associated with high mortality due to an overwhelming inflammatory response. Plasmacytoid dendritic cells (pDC) are potent producers of type I interferons (IFN). This study investigated whether pDC and type I IFN play a role during the early stages of B. pseudomallei infection. Human and murine pDC internalised and killed B. pseudomallei as efficiently as murine conventional DC (cDC). pDC derived from B. pseudomallei-susceptible (BALB/c) mice demonstrated poor intracellular killing and increased IFN-alpha compared to pDC derived from B. pseudomallei-resistant (C57BL/6) mice. This is the first evidence of pDC bactericidal activity against B. pseudomallei infection. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Burkholderia pseudomallei; Melioidosis; Dendritic cells; Interferon-alpha; Interferon-beta
1. Introduction In acute cases of melioidosis, a bacterial infection caused by Burkholderia pseudomallei, patients can rapidly develop fatal septic shock [1]. Characteristics of melioidosis sepsis include significantly increased proinflammatory cytokine expression and the development of multiple organ failure [2]. Investigations of immune cell interactions with B. pseudomallei have found that the macrophage and neutrophil provide important innate defence against B. pseudomallei, while the conventional dendritic cell (cDC) provides a link between the innate and adaptive immune responses [3e10]. To our knowledge, there have been no reports on the role of plasmacytoid DC (pDC) in the immune response toward B. pseudomallei infection.
The capacity to produce large quantities of type I interferons (IFN) is one feature that sets pDC apart from cDC [11]. Historically, pDC and type I IFN were considered ‘antiviral’ mechanisms. It has now been recognised that pDC and type I IFN are capable of eliciting anti-bacterial activity that can be beneficial or detrimental to the host [12e15]. The contribution of pDC to clearance or persistence of B. pseudomallei infection has not been investigated. Therefore, using human and murine pDC, the purpose of this study was to assess the ability of pDC to internalise and kill B. pseudomallei, quantify the type I IFN response of pDC toward B. pseudomallei and determine if B. pseudomallei stimulates phenotypic maturation of pDC.
2. Materials and methods * Corresponding author. Australian Institute of Tropical Health and Medicine, College of Public Health, Medicine and Veterinary Sciences, James Cook University, 1 Solander Road, Townsville, Queensland 4811, Australia. Tel.: þ61 7 4781 6646; fax: þ61 7 4779 1526. E-mail address: [email protected] (N.L. Williams).
2.1. Enrichment of human pDC from peripheral blood Plasmacytoid DC were enriched from peripheral blood mononuclear cells (PBMC), of four healthy male individuals
http://dx.doi.org/10.1016/j.micinf.2014.12.007 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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(aged 28e52), using a pDC isolation kit (Stemcell EasySep™) according to manufacturer's instructions. Prior informed consent was received from all study participants. James Cook University Human Ethics Committee (H4470) provided institutional ethics approval. Purity of enriched pDC (hLMAX (lineage), HLA-DRþ, CD123þ) was confirmed by flow cytometry using a pDC identification kit (IMGENEX, Jomar Bioscience P/L). All flow cytometry was performed using a FACSCalibur with Cell Quest software (BD Biosciences) and post-acquisition analysis was performed using Flow Jo software (Tree Star Inc.). The purity of human pDC enriched from PBMC was 93.9 ± 2.1% (mean ± SEM; data not shown). 2.2. Culture of murine pDC from bone marrow supplemented with FLT-3L Bone marrow (BM) was isolated from C57BL/6 (B. pseudomallei-resistant) and BALB/c (B. pseudomallei-susceptible) mice (n ¼ 3 each, 8e12 week old, James Cook University Small Animal Breeding Facility). James Cook University Animal Ethics Committee (A1601) provided institutional ethics approval. BM cells were cultured at 1 106 cells/ml in pDC media (RPMI-1640 with 10% HIFBS, 1.5 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-Mercaptoethanol and 2.5 mmol/l HEPES buffer) supplemented with FLT-3L (150 ng/ml, Peprotech) using methods previously described [16]. On day 5, 50% of the culture media was replaced. On day 10, pDC expressing PDCA-1 were magnetically selected using biotinylated anti-PDCA-1 antibody (eBioscience) and streptavidin-magnetic nanoparticles (BD Biosciences). Enriched pDC, defined as PDCA-1þ/CD11cþ/B220þ/Ly6Cþ cells, were evaluated by flow cytometry using antimouse CD11c Biotin þ Streptavidin-APC (BD Biosciences), anti-mouse PDCA-1 PE (eBioscience), anti-mouse B220 PerCP-Cy5.5 (BD Biosciences) and anti-mouse Ly-6C FITC (BD Biosciences) [17]. The purity was 91 ± 2.8% for C57BL/6 pDC and 90 ± 3.2% for BALB/c pDC (mean ± SEM; data not shown). 2.3. Culture of murine cDC from BM supplemented with GM-CSF Bone marrow from C57BL/6 and BALB/c mice was also used to culture cDC according to published methods [6]. Isolated BM cells (2 105 cells/ml) were cultured in DC media (RPMI-1640 with 10% HI-FBS, 1.5 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mM 2Mercaptoethanol) supplemented with 10% supernatant from Ag8653 myeloma cells transfected with the gene encoding murine GM-CSF (kindly provided by B. Stockinger, MRC National Institute for Medical Research, Mill Hill, London, UK). On day 3 & 6, 50% of the media was replaced. On day 10 of culture, cDC were harvested.
2.4. B. pseudomallei infection of cells Human pDC (5 104), murine pDC (1 105) and cDC (1 105) were seeded into 96 well plates in pDC media and cultured at 37 C with 5% CO2. Two clinical B. pseudomallei isolates, NCTC 13178 (high virulence) and NCTC 13179 (low virulence; infection of murine pDC and cDC only), were grown to log phase then added to cells at a MOI 1:1 as previously described [6,18]. CpG ODN 2216 (3 mg/ml; SigmaeAldrich) was used as a positive stimulant [19]. After 4 h of culture, internalisation of B. pseudomallei by human pDC, murine pDC and murine cDC was assessed and kanamycin (250 mg/ml; SigmaeAldrich) added to parallel cultures to kill extracellular B. pseudomallei. After 24 h of culture, B. pseudomallei survival within murine pDC and cDC, type I IFN production by human and murine pDC and phenotypic maturation of murine pDC was assessed. 2.5. Assessment of internalisation and intracellular survival of B. pseudomallei Internalisation of B. pseudomallei by human pDC, murine pDC and murine cDC and the intracellular survival of B. pseudomallei within murine pDC and cDC was assessed as previously described [6]. The intracellular bacteria within pDC and cDC were released by lysing the cells with 0.1% Triton-X. Lysates were subsequently diluted and plated in triplicate on Ashdown agar and colonies enumerated after 48 h of culture at 37 C. The percentage of B. pseudomallei internalised at 4 h and the percentage of intracellular B. pseudomallei surviving at 24 h was determined. 2.6. Assessment of type I IFN production by pDC Cell culture supernatants from 24 h uninfected, B. pseudomallei-infected and CpG ODN 2216 -stimulated human and murine pDC were collected and stored at 80 C. The concentration of IFN-a and b in cell culture supernatants was quantified using human and mouse IFN-a (eBioscience) and IFN-b ELISA kits (Sapphire Bioscience) according to manufacturer's instructions. 2.7. Analysis of DC maturation markers on murine pDC Expression of MHC class II and CD86 on 24 h uninfected and B. pseudomallei-infected pDC was analysed by flow cytometry using anti-mouse PDCA-1 Biotin and StreptavidinPerCP (eBioscience), anti-mouse MHC class II PE (BD Biosciences), anti-mouse CD86 PE (BD Biosciences) [20]. The percentage of pDC that were PDCA-1þ/MHCclassIIþ and PDCA-1þ/CD86þ was determined. 2.8. Statistical analysis Statistical analysis of data was performed using GraphPad Prism 6 Software. The production of IFN-a and b by human
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pDC was compared using a 1way ANOVA with Tukey's posthoc multiple comparisons. Murine pDC response to stimulation was tested using 2way ANOVA with post-hoc multiple comparisons to enable comparison between mouse strains and stimulation groups. Differences between tested groups were considered significant if the P value 0.05. 3. Results 3.1. B. pseudomallei internalisation and type I IFN production by human pDC Internalisation of B. pseudomallei (NCTC 13178) by human pDC was evident after 4 h (2 ± 0.67%, mean ± SEM; data not shown). High levels of IFN-a and b were produced by pDC stimulated with CpG ODN 2216 (positive control; Fig. 1). However, B. pseudomallei-infected human pDC demonstrated low IFN-a and b production (Fig. 1). No significant difference was observed in the type I IFN response to B. pseudomallei compared to uninfected human pDC (Fig. 1). 3.2. B. pseudomallei internalisation and intracellular survival within murine pDC and cDC The percentage of B. pseudomallei (NCTC 13178, high virulence) internalised by C57BL/6 pDC was comparable to BALB/c pDC (Fig. 2A). When two B. pseudomallei isolates of contrasting high virulence (NCTC 13178) and low virulence (NCTC 13179) were compared, no significant difference in mean percentage of B. pseudomallei internalised was observed for C57BL/6 pDC (12.6 ± 3.2% and 8.5 ± 1.6% respectively; ±SEM, P ¼ 0.34; data not shown) or for BALB/c pDC (12.2 ± 2.3% and 10.5 ± 1.1% respectively; ±SEM, P ¼ 0.83; data not shown). The internalisation of B. pseudomallei (NCTC 13178) by pDC was also assessed in parallel with murine cDC and found to be comparable (Fig. 2A).
Fig. 1. Type I IFN production by human pDC The production of type I IFN by uninfected, CpG ODN 2216-stimulated and B. pseudomallei einfected (NCTC 13178, high virulence) human pDC was determined by quantifying IFN-a and b concentrations in culture supernatants after 24 h. Human pDC demonstrated high type I IFN production in response to positive stimulation with CpG ODN 2216 but low type I IFN production when infected with B. pseudomallei. Data points represent human pDC isolated from 4 individuals and the line bars depict the mean ± SEM. *P 0.05 and ** P 0.01 determined using a 2way ANOVA with post-hoc multiple comparisons.
Fig. 2. Burkholderia pseudomallei internalisation and intracellular survival within murine pDC and cDC Viable intracellular B. pseudomallei (NCTC 13178, high virulence) within murine pDC and cDC generated from C57BL/6 and BALB/c mice were enumerated at 4 and 24 h of co-culture. The percentage of A) B. pseudomallei internalised at 4 h and B) B. pseudomallei surviving at 24 h was calculated. A) Internalisation of B. pseudomallei was similar in both C57BL/6 and BALB/c pDC. B) The intracellular survival of B. pseudomallei was higher in BALB/c pDC compared to C57BL/6 pDC, though not statistically significant (P ¼ 0.06). This trend was significant for BALB/c cDC compared to C57BL/6 cDC. Bars depict mean ± SEM of three experiments, where three replicate samples were assessed in triplicate. *P 0.05 and NS e not significant determined using a 2way ANOVA with post-hoc multiple comparisons.
The intracellular survival of B. pseudomallei (NCTC 13178) was higher in BALB/c pDC compared to C57BL/6 pDC (Fig. 2B). Both murine pDC and cDC derived from BALB/c mice demonstrated a trend for reduced B. pseudomallei killing compared to pDC and cDC derived from C57BL/6 mice, this trend was statistically significant for BALB/c cDC compared to C57BL/6 cDC. Intracellular survival of B. pseudomallei in BALB/c cDC was also significantly higher than in BALB/c pDC (Fig. 2B). pDC demonstrated similar killing against intracellular B. pseudomallei of high (NCTC 13178) and low virulence (NCTC 13179); C57BL/6 pDC (0.6 ± 0.2% and 6.1 ± 2.6% mean B. pseudomallei survival ± SEM respectively; P ¼ 0.54; data not shown) and BALB/c pDC (6.3 ± 2.1% and 12.2 ± 7.8% mean B. pseudomallei survival ± SEM respectively; P ¼ 0.12; data not shown).
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3.3. Type I IFN production by murine pDC High IFN-a and b was observed for pDC stimulated with CpG ODN 2216 (positive control; Fig. 3A). However, BALB/c pDC demonstrated a reduced capacity to produce IFN-a
(Fig. 3A) and b (data not shown) compared to C57BL/6 pDC. Therefore, the increase in IFN-a/b above the baseline (uninfected pDC) was used for comparisons. The increase in IFN-a by B. pseudomallei-infected BALB/c pDC was significantly higher than that observed for B. pseudomallei-infected C57BL/6 pDC (Fig. 3B). IFN-a production by C57BL/6 and BALB/c pDC was comparable for B. pseudomallei isolates of high (NCTC 13178) and low (NCTC 13179) virulence. Although, the increase in IFN-b produced by C57BL/6 and BALB/c pDC in response to the high virulence B. pseudomallei isolate (NCTC 13178) was comparable, the low virulence B. pseudomallei isolate (NCTC 13179) stimulated significantly less IFN-b production by C57BL/6 pDC (Fig. 3C). 3.4. Murine pDC phenotypic maturation in response to B. pseudomallei To determine if exposure to B. pseudomallei activated phenotypic maturation of pDC, the expression of MHC class II and CD86 on uninfected and B. pseudomallei-infected pDC was compared. MHC class II expression decreased on C57BL/ 6 pDC (1.6 ± 1.5% mean change in MHC IIþ ± SEM) and BALB/c pDC (6.1 ± 3.1% mean change in MHC IIþ ± SEM) infected with B. pseudomallei (NCTC 13178; data not shown). Expression of CD86 increased on C57BL/6 pDC (0.3 ± 3.0% mean change in CD86þ ± SEM) and BALB/c pDC (6.1 ± 1.8% mean change in CD86þ ± SEM) infected with B. pseudomallei (NCTC 1378; data not shown). The changes observed were of a low magnitude and not significant due to high MHC class II and CD86 expression on uninfected murine pDC; 77% and 71% mean MHC IIþ and CD86þ respectively for C57BL/6 pDC, 70% and 72% mean MHC IIþ and CD86þ respectively for BALB/c pDC (data not shown). 4. Discussion
Fig. 3. Type I IFN production by murine pDC To investigate the type I IFN response of murine pDC derived from C57BL/6 and BALB/c mice, the concentration of IFN-a and b in culture supernatants was determined. A) Murine pDC from BALB/c mice, which are known to be poor producers of type I IFN, demonstrated a significantly reduced capacity to produce IFN-a and b (not shown) when stimulated with CpG ODN 2216 for 24 h. Therefore, to compare the type I IFN response of pDC toward B. pseudomallei, the concentration of type I IFN in culture supernatants of uninfected and B. pseudomallei-infected (NCTC 13178, high virulence) murine pDC was quantified and expressed as the fold increase in B) IFN-a and C) IFN-b above baseline (uninfected pDC). B) The increase in IFN-a was significantly higher in B. pseudomallei-infected BALB/c pDC compared to B. pseudomallei-infected C57BL/6 pDC and was not influenced by isolate virulence. C) The production of IFN-b by C57BL/6 pDC was similar to BALB/c pDC in response to NCTC 13178 (high virulence). However, the low virulence B. pseudomallei isolate (NCTC 13179) stimulated significantly less IFN-b production by C57BL/6 pDC. Bars depict mean ± SEM of a representative experiment of three repeat experiments, where three replicate samples were assessed in duplicate. *P 0.05 and **P 0.01 determined using a 2way ANOVA with post-hoc multiple comparisons.
The role of pDC during bacterial infections is underestimated and often thought of as immunomodulating due to conflicting evidence for pDC bactericidal and antigen presenting capacity [21]. This is the first report of internalisation and intracellular killing of B. pseudomallei by both human and murine pDC. The bactericidal activity of murine pDC differed for cells derived from B. pseudomallei-susceptible and resistant hosts. Importantly, murine pDC were as efficient as murine cDC at internalising and killing intracellular B. pseudomallei, providing evidence for the potential of pDC in processing and presentation of B. pseudomallei antigens to T cells. The type I IFN response of pDC varies for different infections and has been implicated in driving an excessive cytokine response that is detrimental to the host [12]. In this study, the production of IFN-a and b by human and murine pDC infected with B. pseudomallei was low in comparison to cytokine levels in CpG ODN 2216 stimulated cultures. Contrasting type I IFN responses by pDC toward bacteria have been reported. Although pDC are capable of producing high
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type I IFN responses to bacteria such as Staphylococcus aureus, other bacteria such as Streptococcus pyogenes, stimulate low type I IFN responses in human pDC similar that observed for B. pseudomallei-infected human pDC [14,24]. Interestingly, B. pseudomallei isolates of high and low virulence induced comparable type I IFN responses. BALB/c mice are known to be poor IFN-a/b producers compared to C57BL/ 6 mice [22]. The increase in IFN-a production by B. pseudomallei-infected BALB/c pDC was significantly higher than that observed for B. pseudomallei-infected C57BL/6 pDC. This trend was observed for pDC infected with both B. pseudomallei isolates of high (NCTC 13178) and low (NCTC 13179) virulence. In contrast, significant differences in IFN-b production between BALB/c and C57BL/6 pDC in response B. pseudomallei was only observed for the low virulence isolate (NCTC 13179). The BALB/c mice are highly susceptible to B. pseudomallei infection and develop disease that parallels the acute form of human melioidosis [3]. The observation that significantly increased IFN-a production correlated with pDC from B. pseudomallei-susceptible mice suggests that the increased IFN-a may be detrimental to the host during B. pseudomallei infection. Following activation, pDC lose their plasmacytoid morphology and acquire morphology similar to cDC. Increased MHC class II and CD86 expression on mature pDC is important for activation of naive T cells [20]. In some cases, pDC maturation responses are exploited to develop an inappropriate immune response that benefits the persistence of bacteria [14]. In this study, B. pseudomallei infection of murine pDC resulted in distinct differences in MHC class II and CD86 expression on BALB/c pDC and C57BL/6 pDC. Unlike cDC which have been shown to significantly increase MHC class II and CD86 expression in response to B. pseudomallei, MHC class II expression was decreased on murine pDC [6]. The magnitude of these changes was masked by high baseline expression on uninfected murine pDC. FLT-3L supplementation for 10 days to improve pDC yield and manipulation of cells to positively select the PDCA-1 positive pDC are likely to have contributed to the elevated baseline expression of MHC class II and CD86 [23]. In summary, this is the first description of the functional responses of human and murine pDC toward B. pseudomallei. We demonstrated that pDC internalised and killed B. pseudomallei as efficiently as cDC. B. pseudomallei virulence did not correlate with an ability to exploit pDC for intracellular survival or affect type I IFN production. Differences in bactericidal activity and IFN-a production were observed for murine pDC generated from B. pseudomallei-susceptible BALB/c mice compared to B. pseudomallei-resistant C57BL/ 6 mice. The BALB/c mouse is a well described animal model of the acute form of human melioidosis [3]. Our findings implicate pDC as an additional cell-mediator contributing to the altered immune responses that underlie the host susceptibility toward B. pseudomallei infection. Further studies on the downstream responses of pDC and type I IFN signalling are required to elucidate their ability to modulate other innate immune cells and to activate B. pseudomallei-specific T cell
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responses during the early phases of B. pseudomallei infection. Conflicts of interest statement The authors declare no conflicts of interest. Acknowledgements We acknowledge the contribution of the study participants who donated their time and blood samples for isolating human plasmacytoid dendritic cells. The Far North Queensland Hospital Foundation and James Cook University supported this study. References [1] Currie BJ, Fisher DA, Howard DM, Burrow JNC, Lo D, Selvanayagam S, et al. Endemic melioidosis in tropical Northern Australia: a 10-year prospective study and review of the literature. Clin Infect Dis 2000;31:981e6. [2] Wiersinga W, Dessing M, Kager P, Cheng A, Limmathurotsakul D, Day N, et al. High-throughput mRNA profiling characterizes the expression of inflammatory molecules in sepsis caused by Burkholderia pseudomallei. Infect Immun 2007;75:3074e9. [3] Leakey AK, Ulett GC, Hirst RG. BALB/c and C57Bl/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis. Microb Pathog 1998;24:269e75. [4] Barnes JL, Williams NL, Ketheesan N. Susceptibility to Burkholderia pseudomallei is associated with host immune responses involving tumor necrosis factor receptor-1 (TNFR1) and TNF receptor-2 (TNFR2). FEMS Immunol Med Microbiol 2008;52:379e88. [5] Woodman ME, Worth RG, Wooten RM. Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils. PLoS ONE 2012;7:e52276. [6] Williams NL, Kloeze E, Govan BL, Korner H, Ketheesan N. Burkholderia pseudomallei enhances maturation of bone marrow-derived dendritic cells. Trans R Soc Trop Med Hyg 2008;102(Suppl 1):S71e5. [7] Charoensap J, Engering A, Utaisincharoen P, van Kooyk Y, Sirisinha S. Activation of human monocyte-derived dendritic cells by Burkholderia pseudomallei does not require binding to the C-type lectin DC-SIGN. Trans R Soc Trop Med Hyg 2008;102(Suppl 1):S76e81. [8] Horton RE, Morrison NA, Beacham IR, Peak IR. Interaction of Burkholderia pseudomallei and Burkholderia thailandensis with human monocyte-derived dendritic cells. J Med Microbiol 2012;61:607e14. [9] Hodgson KA, Morris JL, Feterl ML, Govan BL, Ketheesan N. Altered macrophage function is associated with severe Burkholderia pseudomallei infection in a murine model of type 2 diabetes. Microbes Infect 2011;13:1177e84. [10] Elvin SJ, Healey GD, Westwood A, Knight SC, Eyles JE, Williamson ED. Protection against heterologous Burkholderia pseudomallei strains by dendritic cell immunization. Infect Immun 2006;74:1706e11. [11] Reizis B, Bunin A, Ghosh HS, Lewis KL, Sisirak V. Plasmacytoid dendritic cells: recent progress and open questions. Annu Rev Immunol 2011;29:163e83. [12] Decker T, Muller M, Stockinger S. The yin and yang of type I interferon activity in bacterial infection,. Nat Rev Immunol 2005;5:675e87. [13] Schiavoni G, Mauri C, Carlei D, Belardelli F, Pastoris MC, Proietti E. Type I IFN protects permissive macrophages from Legionella pneumophila infection through an IFN-gamma-independent pathway. J Immunol 2004;173:1266e75.
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